The -2 Mb Prader-Willi/Angelman syndrome (PWS/AS) domain encompasses a group of imprinted genes that are coordinately regulated by a bipartite imprinting center (Ie). The PWS-IC functions to establish and maintain the paternal epigenotype across the domain. The mechanisms by which imprinting is established and maintained in the domain, as well as mechanisms of PWS-IC function, are not weIl-nnderstood. The -3,8 Mb syntenic imprinted region on mouse chromosome 7C serves as a model for PWS and PWS-IC function. The goal of this project is to investigate mechanisms of murine PWS-IC function. Based on DNase hypersensitivity and chromosome conformation capture (3C) studies, we propose that the PWS-IC is composed of multiple regulatory regions that function combinatorially on the paternally-inherited chromosome and engage in long-range interactions within the domain to facilitate imprinting and activate gene expression on the paternal (pat) chromosome. We further postulate that imprinting on the pat chromosome involves factors that bind to the PWS-IC and to loci that interact with the PWS-IC. Thus, we propose 2 Specific Aims: I) We will use 3C and 4C assays for a comprehensive analysis oflong-range interactions involving the PWS-IC on the maternal (mat) and pat chromosomes in different cell types. This should define the spatial organization of the ASIPWS domain on the mat and pat chromosomes, identify regions across the mouse genome that interact with the PWS-IC (and may be involved in the imprinting process), and reveal tissue-specific functions ofthe PWS-IC. This will also allow us to determine if long-range interactions between the PWS-IC and its target genes occur via a looping or tracking mechanism. 2) We will perform a molecular characterization ofthe 6 DNase I hypersensitive (DH) sites we have identified within the PWS-IC on the pat chromosome;we hypothesize these DR sites are the active components of the PWS-IC and together constitute the functional PWS-IC. We also postulate that factors binding within these DH sites facilitate long-range interactions of the PWS-IC and mediate its function. Therefore, we will use various molecular strategies to characterize cis- and trans-acting elements located within these DH sites. These studies should yield new insight into PWS-IC function and provide a strong basis for future genetic analyses of its regulatory components and the sites that interact with it.